Download Assembly and function of AP-3 complexes in cells expressing

Published January 21, 2002
JCB
Article
Assembly and function of AP-3 complexes in cells
expressing mutant subunits
Andrew A. Peden, Rachel E. Rudge, Winnie W.Y. Lui, and Margaret S. Robinson
Department of Clinical Biochemistry, University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK
T
were generated that express 3A, 3B, a 3A2 chimera,
two 3A deletion mutants, and a 3A point mutant lacking a
functional clathrin binding site. All six constructs assembled
into complexes and were recruited onto membranes.
However, only 3A, 3B, and the point mutant gave full
functional rescue, as assayed by LAMP-1 sorting. The
3A2 chimera and the 3A short deletion mutant gave
partial functional rescue, whereas the 3A truncation mutant
gave no functional rescue. These results indicate that the
hinge and/or ear domains of 3 are important for function,
but the clathrin binding site is not needed.
Introduction
Adaptor proteins (APs)* are heterotetrameric complexes that
facilitate cargo selection and coated vesicle budding from
different membrane compartments. Four such complexes
have been identified in mammals, all of which consist of two
large subunits: a subunit and a more divergent, complexspecific subunit (, , , or ); a medium-sized or subunit; and a small or subunit. The subunits are assembled
into a structure resembling Mickey Mouse, with a brick-like
“head,” consisting of the NH2-terminal domains of the two
large subunits, the medium subunit, and the small subunit,
out of which protrude the COOH-terminal “ear” domains
of the large subunits, connected by flexible hinges. The AP-1
and AP-2 complexes were identified first as abundant components of clathrin-coated vesicles. More recently, the AP-3
and AP-4 complexes were identified, primarily by finding
homologues of the AP-1 and AP-2 subunits in the EST
Address correspondence to Margaret S. Robinson, University of Cambridge, CIMR, Wellcome Trust/MRC Building, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2XY, UK. Tel.: (44) 1223-330163. Fax:
(44) 1223-762640. E-mail: [email protected]
Andrew A. Peden and Rachel E. Rudge contributed equally to this paper.
Andrew A. Peden’s present address is Howard Hughes Medical Institute,
Department of Molecular and Cellular Physiology, Stanford University
School of Medicine, CA 94305-5426.
*Abbreviations used in this paper: AP, adaptor proteins; HPS, Hermansky Pudlak syndrome.
Key words: coated vesicles; adaptors; clathrin; mocha; pearl
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The Journal of Cell Biology, Volume 156, Number 2, January 21, 2002 327–336
http://www.jcb.org/cgi/doi/10.1083/jcb.200107140
database, raising antibodies against recombinant proteins,
and showing by coimmunoprecipitation experiments that
the proteins were assembled into two novel complexes (Robinson and Bonifacino, 2001).
Although the AP-3 complex was identified relatively recently, it has been much more amenable to genetic analysis
than the AP-1 or AP-2 complexes. When the subunit of
the complex was first cloned, it was found to be the mammalian homologue of the protein encoded in Drosophila by
the garnet gene, one of the classical eye color genes (Ooi et
al., 1997; Simpson et al., 1997). garnet mutants have defective pigment granules, and because of similarities between
pigment granules and lysosomes, this suggested a role for the
AP-3 complex in the trafficking of proteins destined for
lysosomes and related organelles. Subsequent studies in yeast
showed that deleting any of the four AP-3 subunit genes
causes missorting of a subset of proteins normally destined
for the vacuole, the yeast equivalent of the lysosome (Cowles
et al., 1997; Stepp et al., 1997). Two naturally occurring
mouse mutants have also been identified with mutations in
AP-3 subunits. The mocha (mh) mouse has a null mutation
in the subunit of the complex (Kantheti et al., 1998),
whereas the pearl (pe) mouse has effectively a null mutation
in the 3A subunit (small amounts of mRNA can be detected which encode a trunctated protein, missing the last
125 amino acids, but the protein has not been detected on
Western blots) (Feng et al., 1999; Zhen et al., 1999). The
mouse mutants have a similar phenotype to the human
327
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he mouse mutants mocha and pearl are deficient in
the AP-3 and 3A subunits, respectively. We have
used cells from these mice to investigate both the
assembly of AP-3 complexes and AP-3 function. In mocha
cells, the 3 and 3 subunits coassemble into a heterodimer,
whereas the 3 subunit remains monomeric. In pearl cells,
the and 3 subunits coassemble into a heterodimer,
whereas 3 gets destroyed. The yeast two hybrid system was
used to confirm these interactions, and also to demonstrate
that the A (ubiquitous) and B (neuronal-specific) isoforms
of 3 and 3 can interact with each other. Pearl cell lines
Published January 21, 2002
328 The Journal of Cell Biology | Volume 156, Number 2, 2002
Figure 1. AP-3 subunits in mh and pe cells. (a) Western blots of lysates from the mh and pe cells, as well as a wild-type control cell line
(melan-a cells), were probed with antibodies against all four AP-3 subunits and with an antibody against the AP-1 subunit as a control. Only
the 3 subunit is detectable in the mh cells under these conditions, while only the and 3 subunits are detectable in the pe cells. (b–d) The
same cells were labeled for immunofluorescence with an antibody against the AP-3 subunit. Control cells (b) have the typical punctate pattern,
mh cells (c) show no labeling, and pe cells (d) have diffuse cytoplasmic labeling, indicating that although the subunit is detectable, it is
cytosolic rather than membrane-associated. Bar, 20 m.
obligatory coupling between AP-3 and clathrin in mammalian cells has not yet been resolved.
Here we have taken advantage of the mouse mutants to
address two questions. First, what happens to the other AP-3
subunits if one of them is missing? And second, to what extent can we rescue the phenotype of mocha and pearl cells
by transfecting them with either a wild-type copy of the
missing subunit, or alternatively with one that has been altered in some manner? In particular, are 3A subunits that
no longer have the clathrin binding motif still functional?
These studies provide insights into the assembly and function not only of the AP-3 complex, but of the other AP
complexes as well.
Results
Assembly of AP-3 complexes in mh and pe cells
To investigate the mocha (mh) and pearl (pe) phenotypes at
the cellular level, we have established an mh cell line and
made use of a previously established pe cell line. Fig. 1 a
shows Western blots of extracts from these cells probed for
the four AP-3 subunits: , 3, 3, and 3. In the cells from
the mocha homozygote (mh/mh), , 3, and 3 are all undetectable, while 3 is detectable but at reduced levels. In the
pearl cells (pe/pe), 3 and 3 are both undetectable, while and 3 are both detectable at reduced levels. When the cells
are labeled for immunofluorescence using a -specific antibody, the typical punctate pattern can be seen in control cells
(), the mh cells are unlabeled, and the pe cells show diffuse
cytoplasmic labeling, indicating that although is present in
pe cells, it is unable to associate with membranes in the absence of the 3 and/or 3 subunits (Fig. 1, b–d).
Previous studies have shown that in mh and pe mice, mRNAs
encoding the three nonmutated subunits are expressed
normally, indicating that the subunits are synthesized at
wild-type levels but then get degraded because they are unstable (Kantheti et al., 1998; Feng et al., 1999). Thus, trace
amounts of the nonmutated subunits may be present in the
mh and pe cells, which are below the limit of detection on
whole cell Western blots. To try to concentrate the subunits,
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genetic disorder Hermansky Pudlak syndrome (HPS), and
indeed a subset of HPS patients has been identified with
mutations in 3A (Dell’Angelica et al., 1999). In both the
mice and the humans, AP-3 deficiency results in hypopigmentation because of abnormalities in melanosomes, prolonged bleeding because of deficiencies in platelet dense
granules, and reduced secretion of lysosomal enzymes into
the urine. AP-3–deficient fibroblasts appear relatively normal, but several studies have revealed that lysosomal membrane proteins in these cells show increased trafficking via
the plasma membrane (Dell’Angelica et al., 1999, 2000; Le
Borgne et al., 1998). The mh mouse also has neurological
defects, although the pe mouse and the 3A-deficient humans are neurologically normal. This is presumably because
there is a second 3 isoform, 3B, which is specifically expressed in neurons and neuroendocrine cells (Kantheti et al.,
1998). Together, these findings indicate that the AP-3 complex facilitates the trafficking of certain types of cargo to lysosomes and lysosome-related organelles.
Whether or not AP-3 is associated with clathrin is still
somewhat controversial. Unlike AP-1 and AP-2, AP-3 is not
enriched in purified clathrin-coated vesicles (Newman et al.,
1995; Simpson et al., 1996). However, a clathrin-binding
consensus sequence, L[L,I][D,E,N][L,F][D,E], has been
identified in the hinge domains of 1 and 2, and similar
sequences are found in 3A and 3B which can interact
with clathrin in vitro (Dell’Angelica et al., 1998; ter Haar et
al., 2000). AP-3 is also able to coassemble with clathrin in
vitro and to support clathrin recruitment onto liposomes
(Dell’Angelica et al., 1998; Drake et al., 2000). On the other
hand, an in vitro system for the budding of synaptic-like microvesicles from endosomes has been shown to require AP-3
but not clathrin (Shi et al., 1998), indicating that the two
can function independently. In addition, gene deletion studies in yeast indicate that AP-3 and clathrin function on different pathways; however, yeast 3 does not contain a clathrin binding motif. Immunolocalization studies, at both the
light and the electron microscope level, have yielded conflicting results (Simpson et al., 1996, 1997; Dell’Angelica et
al., 1998). Thus, the question of whether or not there is an
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AP-3 assembly and function | Peden et al.
329
tracts from pe spleen under nondenaturing conditions with a
3A-specific antibody (the reason for using spleen for this
experiment is that it is possible to get a more highly concentrated extract from tissues than from cultured cells, and
AP-3 is known to be expressed at particularly high levels in
spleen). Fig. 2 (right) demonstrates that there are indeed
small amounts of truncated 3A made in pe cells which can
coassemble with the other three subunits to form a heterotetramer, although we cannot rule out the possibility that
some of the 3 may also be assembled into a heterotrimer.
Together, these studies indicate that 3 and 3 can interact
with each other, forming heterodimers in mh cells, and that
and 3 also interact with each other, forming heterodimers in pe cells. The interactions that we can detect in
the mutant cells are shown diagrammatically in the bottom
part of Fig. 2.
and to determine whether they are associated with each
other, we immunoprecipitated cell extracts with antibodies
against the , 3, and 3 subunits under nondenaturing
conditions, and with anti-3, which only recognizes the denatured protein, in samples that had been denatured by boiling in SDS. Western blots of the immunoprecipitates were
then probed with antibodies against all four of the AP-3 subunits, as well as with an antibody against the subunit of
AP-1 as a control.
Fig. 2 (left) shows that under these conditions both 3
and 3 can be detected in the mh cells, and furthermore that
they assemble into a heterodimer since both subunits come
down with anti-3. In contrast, the 3 in these cells appears
to exist as a monomer, since it does not coimmunoprecipitate with any of the other subunits. In the pe cells (Fig. 2,
middle), and 3 form heterodimers which come down
with antibodies against both subunits, and in addition, a
small amount of 3 can be detected which coprecipitates
with the /3 dimers. This suggests either that the 3 may
be assembling with the and 3 subunits into a heterotrimer, or alternatively, since the pe mutation is not a true null,
that there are small amounts of truncated 3A expressed in
these cells which can form heterotetramers. To distinguish
between these two possibilities, we immunoprecipitated ex-
Sorting of lysosomal membrane proteins
Previous studies have shown that the steady-state distribution of lysosomal membrane proteins, such as LAMP-1,
LIMP-2, and CD63, appears normal in AP-3–deficient
cells, but that they follow different trafficking routes to the
lysosome. Le Borgne et al. (1998) demonstrated that cells
that had been depleted of 3 by incubation with antisense
oligonucleotides showed increased endocytosis of antibodies
against LAMP-1 and LIMP-2 that had been added to the
culture medium, indicating that these proteins had been rerouted to the cell surface. Dell’Angelica et al. (1999, 2000)
reported similar findings on primary cultures of fibroblasts
from 3A-deficient patients and from mh and pe mice. We
have also found that antibodies against LAMP-1 are readily
endocytosed by both the mh and the pe cell lines (see Figs. 4
and 8). However, because all cell lines are different from
each other, we needed to confirm that the antibody endocytosis was a direct result of the AP-3 deficiency. Thus, we
transiently transfected the cells with a wild-type copy of the
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Figure 2. Immunoprecipitates and Western blots of AP-3 subunits
from mh and pe cells. Extracts of cultured mh cells (left), cultured
pe cells (middle), and spleens from both pe and control mice (right)
were immunoprecipitated with the indicated antibody. Most immunoprecipitations were performed under nondenaturing conditions,
with the exception of the anti-3 immunoprecipitations, which
were performed in the presence of SDS. The gels were then blotted
onto nitrocellulose and probed with the indicated antibody. An
antibody against the AP-1 subunit was included as a control. The
mh cells can be seen to express trace amounts of 3 and 3, which
form a dimer, whereas the 3 subunit appears to be monomeric.
The and 3 expressed in the pe cells also form a dimer, and in
addition a small amount of complete heterotetramer is made. The
various subcomplexes that form in the mh and pe cells are indicated
diagrammatically.
Subunit interactions
To investigate interactions between the AP-3 subunits further, and also to determine whether the A and B isoforms of
the various subunits can associate with each other, we cloned
the cDNAs into the vectors pGBT9 and pGAD424 for analysis using the yeast two-hybrid system. Fig. 3, a and b, shows
that interacts under these conditions with 3A and 3B,
both of which are expressed ubiquitously. In addition, 3A
interacts both with the ubiquitously expressed 3A and with
the neuronal-specific 3B (Fig. 3 c), and 3B interacts with
3A (Fig. 3 d). For reasons that are not clear, we were unable to detect any interaction between 3B and 3B (unpublished data). However, the observations that 3A can interact with 3B and that 3B can interact with 3A suggest
that, instead of having a neuronal-specific complex and a
nonneuronal-specific complex, the subunits can “mix and
match” if they are coexpressed in the same cell. The interactions involving the AP-3 subunits appear to be complex-specific, as we could detect no interactions between 3 and 1
(Fig. 3 c), or between and 1 (unpublished data). Other
pairs of subunits were also tested, but the only interactions
we were able to detect were those shown in Fig. 3.
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330 The Journal of Cell Biology | Volume 156, Number 2, 2002
Figure 3. Subunit interactions between AP-3 subunits detected using the yeast two-hybrid system. Yeast cells were transformed with cDNAs
in either pGBT9 or pGAD424, as indicated, and interactions were assayed by growth in the absence of histidine. Panels a and b show that the
subunit interacts with both 3A and 3B, panel c shows that the 3A subunit interacts with both 3A and 3B, and panel d shows that the
3B subunit interacts with 3A.
Introduction of altered subunits
The availability of an assay for AP-3–mediated sorting
means that we can now test whether not only wild-type subunits, but also subunits that have been altered in some way,
are functional. Because some such subunits might be expected to give intermediate rescue, we developed a quantitative assay based on FACS analysis for use on stably transfected cells (see below). Unfortunately, we have so far been
unable to obtain any mh cell lines that were homogeneously
expressing ; thus, we decided to concentrate on pe cells. In
addition to cells expressing wild-type 3A, we have established pe cell lines expressing 3B, a 3A-2 chimera containing the 3A NH2-terminal domain (amino acids 1–686)
Figure 4. Phenotype of rescued and
nonrescued mh cells. mh cells were
transiently transfected with wild-type ,
then incubated with a mixture of rat
anti–LAMP-1(b) and fluorescent WGA
(c) for 6 h. Triple labeling shows that the
cell in the field that is expressing has
taken up less anti–LAMP-1 than its
neighbors, indicating that there is less
misrouting of LAMP-1 to the plasma
membrane in this cell. Uptake of WGA
is similar in all of the cells. Bar, 20 m.
but the 2 hinge and ear domains (3A2); two 3A deletion mutants (3A
807–831 and 3A807 stop); and a 3A
point mutant (3A817AAA). The 3A
807–831 mutant is
missing a fragment of the distal part of the hinge, including the clathrin binding domain (amino acids 817–822),
whereas the 3A807stop mutant is missing the distal part of
the hinge, including the clathrin binding domain, and all of
the ear. In the 3A817AAA mutant, the clathrin binding
motif has been mutated from SLLDLD to AAADLD. This
same mutation has been shown to abolish clathrin binding
in vitro (Dell’Angelica et al., 1998; unpublished data). Schematic diagrams of the constructs are shown in Fig. 5 a.
We first investigated whether the constructs were capable of
assembling into AP-3 complexes by immunoprecipitating cell
extracts with an antibody against the subunit, then probing
Western blots of the immunoprecipitates with antibodies
against the other three subunits. Fig. 5 b shows that in the
nontransfected cells, only 3 coprecipitated with at detectable levels (see also Figs. 1 and 2), while in cells transfected
with 3A, all three of the other subunits were detectable, indicating that the 3A had assembled into complexes together
with 3, thus protecting the 3 subunit from degradation.
Similarly, in cells transfected with the other five constructs,
the construct itself and the 3 subunit came down with the and 3 subunits. These results are consistent with studies on
the AP-1 and AP-2 complexes, which indicate that the NH2terminal domains of the subunits are incorporated into the
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defective subunit. Cells transfected with both the and the
3A constructs could be identified with a -specific antibody: the nontransfected mh cells were unlabeled, whereas
the nontransfected pe cells gave diffuse rather than punctate
labeling with the anti- antibody (see Fig. 1). Fig. 4 shows
transiently transfected mh cells labeled for (a), endocytosed
anti-LAMP-1 (b), and endocytosed WGA (c). The cell expressing has taken up significantly less anti–LAMP-1 than
its nontransfected neighbors, while all of the cells have taken
up similar amounts of WGA, indicating that endocytosis in
general is not impaired in the -expressing cell. Similar results were obtained with pe cells transfected with 3A (see
Fig. 8 a).
Published January 21, 2002
AP-3 assembly and function | Peden et al.
331
Figure 5. (a) Schematic diagrams of six
3-based constructs that were stably
transfected into pe cells. (b) Extracts from
cell lines expressing each of the six
constructs, as well as a cell line stably
transfected with empty vector, were
immunoprecipitated with anti-, and blots
were probed with antibodies against , the
construct itself (in most cases anti-3A,
although the last two lanes were probed
with antibodies against 3B and 2,
respectively), 3, and 3. Only and 3
can be detected in the immunoprecipitate
from the cell line transfected with the
empty vector, whereas all four subunits
can be detected in the cell lines expressing
the six constructs, indicating that complete
heterotetramers have been formed.
Figure 6. Immunofluorescence of cell lines expressing each of the
six constructs labeled with anti-. All of the cells show punctate
labeling with some concentration in the perinuclear region, indicating
that the AP-3 complexes assembled from all six constructs are
recruited onto membranes. Bar, 20 m.
is green and clathrin is red). In cells expressing wild-type
3A, AP-3 and clathrin were found to have distinct distributions: (a) they are often in the same general vicinity, and in
some cases there may be overlap (yellow), but much of the
AP-3 labeling is negative for clathrin. In contrast, when cells
are double labeled for the AP-1 adaptor complex and clathrin
(b), essentially all of the structures that are positive for AP-1
Figure 7. Confocal micrographs showing double labeling for AP
complexes and clathrin. pe cells expressing 3A (a), 3A2 (c), or
3A817AAA were double labeled with anti- (green) and anti-clathrin
(red), and COS cells were double labeled with anti- (green) and
anti-clathrin (red). Essentially all of the anti- labeling is also positive
for clathrin (note how there are no green dots in b, only yellow). In
contrast, in all three cell lines expressing 3 constructs, much of the
AP-3 labeling is negative for clathrin. (Transfected pe cells could
not be double labeled with anti- and anti-clathrin because the
anti- mAb only recognizes the protein in nonrodent cells.) Bar: (a
and b) 10 m; (c and d) 20 m.
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“head,” where they can interact with the other subunits, while
the hinge and ear domains have other functions. The 3 subunit in all of the immunoprecipitates is presumably 3A,
since 3B is only expressed in neurons and neuroendocrine
cells. Thus, this result is consistent with results from the twohybrid experiments, which indicate that although 3B is normally only expressed in the same cells that express 3B, it can
interact with the ubiquitously expressed 3A.
To determine whether the complexes could be recruited
onto membranes, cells expressing each of the six constructs
were labeled for immunofluorescence with anti-. In nontransfected pe cells, has a diffuse cytoplasmic distribution
(Fig. 1 d); however, in cells expressing each of the five constructs, the labeling was punctate, indicating that it was associated with membranes (Fig. 6). We also compared the distribution of AP-3 in cells expressing the various constructs
with that of clathrin, using a confocal microscope (Fig. 7; AP-3
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332 The Journal of Cell Biology | Volume 156, Number 2, 2002
(green) are also positive for clathrin (red; note that there is extensive yellow labeling and very little green). In cells expressing the various constructs, the extent of overlap between AP-3
and clathrin was very similar to that in cells expressing wildtype 3A. This is in spite of the fact that the 3A2 chimera
(c) has more potential clathrin binding sites than wild-type
3A (not only the clathrin-binding consensus sequence
LLNLD in its hinge domain, but also three copies of another
motif implicated in clathrin binding, DLL [Morgan et al.,
2000], as well as clathrin binding activity in its ear domain
[Owen et al., 2000]). Constructs lacking a clathrin binding
domain, like 3A817AAA (d), did not look appreciably different from the other constructs: again, there were some yellow structures, as well as red and green. However, at the light
microscope level, it is impossible to resolve two structures that
are less than 0.2 m apart from each other, so to quantify
the true extent of overlap between AP-3 and clathrin in cells
expressing the various constructs, it will be necessary to go to
the electron microscope level.
Functional rescue of pe cells
Having established that all six constructs are able to coassemble with the other AP-3 subunits and be recruited onto
membranes, we went on to investigate their ability to rescue
the LAMP-1 missorting phenotype. For these experiments, a
rat monoclonal antibody against LAMP-1 was directly conjugated to Alexa Fluor 488. Double-labeling immunofluorescence performed on a mixture of pe cells transfected with
empty vector and pe cells expressing wild-type 3A, incubated with the antibody for 6 h, showed that the nonexpressing cells took up significantly more antibody than the
expressing cells (Fig. 8 a).
For quantitative studies, each stably transfected cell line
was incubated for 6 h either with the anti–LAMP-1 antibody or with an isotype control antibody (anti-KLH), which
also had been conjugated to Alexa Fluor 488. The cells were
then trypsinized, fixed, and analyzed by flow cytometry. Fig.
8 b shows the results obtained with one of the cell lines expressing wild-type 3A. The peak marked “control” shows
the fluorescence intensity of the 3A-expressing cells incubated with the control antibody, which is likely to be taken
up by fluid phase endocytosis only. The peaks marked
“3A” and “empty vector” show the levels of fluorescence
intensity observed when the 3A-expressing line and one of
the cell lines transfected with empty vector were incubated
with the anti–LAMP-1 antibody. In both cases, the fluores-
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Figure 8. Functional rescue of pe cells
by the various 3 constructs. (a) A mixture
of cells transfected with empty vector
and cells expressing 3A were incubated
6 h with anti–LAMP-1 directly conjugated
to Alexa Fluor 488, then double labeled
with anti-. 3A-expressing cells
(arrow), which could be identified by the
punctate, perinuclear distribution of ,
took up less anti–LAMP-1 than cells
transfected with empty vector. (b)
Anti–LAMP-1 uptake analyzed by flow
cytometry. The control peak shows the
fluorescence intensity of a 3A-expressing
cell line incubated with a control antibody (anti-KLH) conjugated to Alexa
Fluor 488. The empty-vector peak shows
the level of fluorescence intensity when
a nonexpressing pe cell line was incubated with the anti–LAMP-1 antibody,
whereas the 3A peak shows the level of
fluorescence intensity when the pe cell
line expressing 3A was incubated with
the anti–LAMP-1 antibody. Anti–LAMP-1
uptake in the 3A-expressing cell line
was 27% that of the empty vector–
transfected line. (c) Graph of the results
obtained by flow cytometry. In each
case three different cell lines, stably
expressing each of the six constructs
were analyzed, as well as three cell lines
transfected with empty vector. In each
experiment, 10,000 cells were counted
and the experiments were repeated on
different days and the results for each
cell line were averaged. The values were
obtained by dividing the geometric
mean fluorescent intensity of cells incubated with the anti–LAMP-1 antibody by that of cells incubated with the control antibody. Error bars
show the standard deviation from the mean. 3A, 3B, and 3A817AAA constructs all gave good rescue (P 0.0001); 3A2 and
3A
807–831 both gave partial rescue (P 0.0059 and P 0.0015, respectively); and 3A807 stop gave no significant rescue (P 0.2523).
P values were obtained using a one-tailed unpaired t test. Bar, 20 m.
Published January 21, 2002
AP-3 assembly and function | Peden et al.
Discussion
The availability of mice with null, or effectively null, mutations in AP-3 subunits has enabled us to study AP-3 assembly and function in mammalian cells. In the past we and
others have transfected mammalian cells with mutant AP-1
and AP-2 subunits for studies on both assembly and function, but interpretation of these studies has been complicated by the presence of endogenous wild-type subunits
(Robinson, 1993; Page and Robinson, 1995). In the present
study, we have been able to introduce mutant subunits into
cells with little or no background.
We first investigated whether partial AP-3 complexes were
able to assemble in the absence of a particular subunit. We
found that mh cells, missing the subunit, assembled heterodimers from the 3 and 3 subunits, whereas the 3
subunit remained monomeric. The pe cells, which express
very low levels of truncated 3A, mainly assembled heterodimers consisting of and 3 subunits, as well as a very
small amount of complete heterotetramer. All of the subunits were destabilized to some extent by the absence of either or 3, but this was particularly striking in the case of
the 3 subunit, which seemed to exist only as a dimer with
the 3 subunit, even when the 3 subunit was barely detectable.
In addition to the naturally occurring AP-3 mouse mutants, knockout mice have been generated which lack the
AP-1 and 1A subunits. Both mouse strains die during
embryogenesis; however, a fibroblast line has been established from the 1A knockout mice, and these cells were
found to assemble a heterotrimer consisting of , 1, and
1 subunits. This complex appears to be fairly stable, although (like the /3 heterodimer) it is unable to be recruited onto membranes (Meyer et al., 2000). The knockout mouse dies so early that it has not been possible to
establish a null cell line, but cells from the heterozygote
have been examined and found to express the subunit at
50% normal levels. Both 1 and 1 completely comigrate
with by gel filtration in these cells, indicating that any excess 1 and 1 are unstable and get degraded (Zizioli et al.,
1999). Similarly, our own studies show that in the absence
of the subunit, the stability of 3 and 3 is greatly reduced. However, somewhat different findings have been reported in yeast. When the AP-3 , 3, or 3 subunits are
deleted, the stability of 3 does not appear to be affected, although it fractionates differently by gel filtration, in particular in the 3-disrupted strain, where it appears to be monomeric (Panek et al., 1997). In contrast, deleting the -like
subunit of the AP-2–related complex in yeast does not affect
the 2 subunit, but it has a profound effect on the stability
of 2 (Yeung et al., 1999). Thus, it is difficult to predict
how a particular AP subunit will behave in a particular organism; however, all of these studies consistently show that
there are strong interactions between the /// subunits
and the subunits, and between the and subunits.
These same interactions can be detected using the yeast
two-hybrid system. The interactions that we report between
the and 3 subunits, and between the 3 and 3 subunits, are consistent with interactions that we and others
have reported between the corresponding pairs of subunits
in the AP-1, AP-2, and AP-4 complexes (Page and Robin-
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cence intensity is higher than that observed with the control
antibody, indicating that the anti–LAMP-1 has been taken
up by receptor-mediated endocytosis as well as fluid-phase
endocytosis. However, the fluorescence intensity of the
3A-expressing line is 4.41 times that of the control. In contrast, the fluorescence intensity of the cell line transfected
with empty vector is 16.61 times that of its own control (unpublished data, but similar results were obtained with all of
the cell lines incubated with the control antibody). Thus,
the amount of specific uptake of anti–LAMP-1 in the 3Aexpressing line is 27% of that in the empty vector–transfected line. This is comparable to results obtained by others
who have investigated anti–LAMP-1 uptake in normal and
AP-3-deficient primary fibroblasts from both mice and humans: wild-type cells were reported to internalize between
20 and 45% as much antibody as mutant cells (Dell’Angelica et al., 1999, 2000).
Fig. 8 c shows the extent of functional rescue obtained
with all six constructs. In each case, three different cell lines
stably expressing each of the six constructs were analyzed, as
well as three cell lines transfected with empty vector. The experiments were repeated on different days and the results for
each cell line were averaged. 3A can be seen to give the best
rescue, with anti–LAMP-1 uptake 4.89 0.237 (SD), as
compared with 17.25 0.821 in cells transfected with
empty vector. The 3B construct also rescued well, with
anti–LAMP-1 uptake 6.58 0.421. Whether the difference
between the 3A- and 3B-expressing cells reflects functional differences between the two 3 isoforms, or whether
it reflects other differences between the various cell lines
(since wild-type cell lines can vary at least this much in their
anti-LAMP-1 uptake), is not at present clear. The 3A2
chimera and the 3A
807–831 deletion mutant both partially rescued the missorting phenotype, with anti–LAMP-1
uptake 10.30 2.620 and 10.48 1.635 respectively.
However, there was no significant difference in antibody
uptake between cells expressing the truncation mutant,
3A807stop (15.73 3.512), and cells transfected with
empty vector. Thus, the distal hinge and/or ear domain of
the 3 subunit appears to be required for AP-3 function,
even though the construct lacking these domains is still able
to assemble into AP-3 complexes and be recruited onto
membranes.
There are at least two possible explanations for the intermediate level of functional rescue that we observed with the
3A
807–831 mutant. One possibility is that the clathrinbinding consensus sequence, which is missing from this construct, is required for full functional rescue. Alternatively, the
construct might be defective in some other way: for instance,
the ear might not be correctly folded. The point mutant
3A817AAA enables us to distinguish between these two possibilities. Instead of a 25-amino acid deletion, this construct
has only 3 amino acid substitutions, but (at least in vitro) it is
unable to bind clathrin (Dell’Angelica et al., 1998). Fig. 8 c
shows that when this construct is expressed in living cells, anti–
LAMP-1 uptake is 7.49 1.128, which is only slightly more
than in 3A-expressing cells and not significantly different
from 3B-expressing cells. Thus, although the hinge and/or
ear domains of 3A are needed for protein function, the clathrin-binding consensus sequence appears to be dispensable.
333
Published January 21, 2002
334 The Journal of Cell Biology | Volume 156, Number 2, 2002
could simply be due to variability from one cell line to another (Dell’Angelica et al., 1999, 2000). However, although
3B can functionally substitute for 3A, the opposite may
not be true. Synaptic-like microvesicle budding from endosomes has been shown to be dependent upon brain AP-3;
liver AP-3 will not suffice (Faundez et al., 1998). This suggests that the neuronal-specific isoform of either 3 or 3,
or both, is required for this activity. Brain AP-3 contains a
mixture of A and B isoforms of both 3 and 3, while the
stably transfected cells express only 3B and 3A; thus, it
should be possible to test the relative importance of neuronal-specific 3 and 3 by determining whether AP-3
complexes from the transfected cells will substitute for brain
AP-3 in the in vitro budding assay.
Two of our constructs gave partial rescue: the 3A2 chimera and the deletion mutant 3A
807–831. In contrast,
the premature truncation mutant 3A807stop was unable
to rescue even partially. This finding indicates that the distal
hinge and/or ear domains of 3 are essential for function. In
the 3A
807–831 mutant, part of the distal hinge is missing, and this fragment may either contribute directly to 3
function, or alternatively may aid in the folding of the ear.
Virtually nothing is known about the ear domain of 3, although the 2 ear has been shown to bind both clathrin and
certain coated vesicle accessory proteins, including AP180,
epsin, and Eps15 (Owen et al., 2000). We have shown that
AP-3 complexes containing truncated 3A are recruited
onto the membrane, and presumably they are able to select
cargo since they contain 3A (Ohno et al., 1998). Thus, the
lack of functional rescue suggests that they fail to produce
coated vesicles, consistent with an inability to recruit structural or accessory proteins that might be essential for this
event. The ability of the 3A2 construct to give partial rescue suggests that the 3 and 2 ears may recruit some of the
same proteins. However, gels of GST pulldowns using the
3A ear showed no obvious candidate binding partners, and
clathrin could not be detected on Western blots of the pulldowns (D. Owen, personal communication; unpublished
data). Thus, the precise function of the 3 ear remains elusive. However, its importance is highlighted by its evolutionary conservation: the 3 ear domains of both Drosophila
and C. elegans are significantly homologous to the ear domains of mammalian 3A and 3B, although both S. cerevisiae and S. pombe 3 subunits appear to lack any ear domain.
Yeast 3 subunits also lack a clathrin-binding consensus
sequence, but both C. elegans and Drosophila have such sequences in their 3 distal hinge domains (LIDVD and
LLDLD, respectively). However, we show here that it is possible to mutate this sequence to one that can no longer bind
clathrin in vitro (Dell’Angelica et al., 1998), and yet still get
functional rescue. This result can now be added to the
weight of evidence against an obligatory coupling between
AP-3 and clathrin. At present we cannot rule out the possibility that there might be other clathrin binding sites in
AP-3, even though there are no obvious candidates (we have
tested the hinge and ear domains of the subunit, but are
unable to detect any clathrin in GST pulldowns [unpublished data]). Double labeling for AP-3 and clathrin at the
electron microscope level should help to determine whether
Downloaded from on June 18, 2017
son, 1995; Aguilar et al., 1997; Hirst et al., 1999; Takatsu et
al., 2001). In addition, in a previous study using the yeast
two-hybrid system, we were able to demonstrate interactions
between the two large subunits of the AP-1 and AP-2 complexes, / and (Page and Robinson, 1995). However, interactions between the large subunits appear to be more difficult to reconstitute, and so far we have been unable to
demonstrate any interactions between and 3, or between
the and 4 subunits of the AP-4 complex (Hirst et al.,
1999). and 4 have been shown recently to interact in the
presence of 4 using the yeast three-hybrid system, however
(Takatsu et al., 2001), so it is possible that 3 may promote
the interaction between and 3. One observation that supports the notion that and 3 interact in vivo is the finding
that mh cells have greatly reduced levels of the 3 and 3
subunits. This suggests that 3 may be unstable in the absence of , which in turn would destabilize 3.
We also used the yeast two-hybrid system to investigate
whether the ubiquitously expressed (A) and neuronal-specific (B) 3 and 3 subunits were capable of interacting
with each other. Interactions were detected between 3A
and 3A, between 3A and 3B, and between 3B and
3A, although surprisingly not between 3B and 3B.
The ability of the A and B isoforms to interact under more
physiological conditions was demonstrated in our transfection experiments. When pe cells were transfected with 3B,
they formed heterotetramers containing the ubiquitously
expressed 3A subunit. Heterotetramers were also assembled from the other five constructs, as expected since they
all contained the 3A NH2-terminal domain. Perhaps less
predictable was the ability of all the constructs to be recruited equally well onto membranes. Although studies
from our own lab and others indicate that the COOH-terminal hinge and ear domains of the adaptor large subunits
are not absolutely essential for membrane association,
AP complexes containing earless or are compromised
in their ability to bind to their respective membranes
(Robinson, 1993); however, the earless 3A construct,
3A807stop, showed no increased cytosolic background.
We were also interested in the extent to which the various
complexes colocalized with clathrin, in particular complexes containing the 3A2 chimera, since multiple clathrin binding sites have been identified and/or predicted in
the 2 COOH-terminal hinge and ear domains (Dell’Angelica et al., 1998; Morgan et al., 2000; Owen et al., 2000).
However, this construct, like the others, was at least partially localized to structures that were negative for clathrin.
In contrast, AP-1 and AP-2 both show essentially complete
colocalization with clathrin (Fig. 7; Robinson, 1987), indicating that the presence of the 2 hinge and ear domains is
not enough for a stable association with clathrin, and that
there must be additional interactions, possibly involving
the and subunits (Goodman and Keen, 1995; Morgan
et al., 2000; Doray and Kornfeld, 2001).
Although all of the constructs assembled into complexes
and were recruited onto membranes equally well, they varied
widely in their ability to rescue the missorting phenotype of
the pe cells. Using a quantitative assay for LAMP-1 mislocalization, we were able to show that 3B gave nearly as good
functional rescue as 3A; indeed, the differences that we saw
Published January 21, 2002
AP-3 assembly and function | Peden et al.
there is any colocalization between the two in cells expressing 3 with a mutated clathrin binding domain. However,
much of the evidence in support of an association between
AP-3 and clathrin comes from studies on the LLDLD sequence, and the present study demonstrates that this sequence is not essential for AP-3 function in vivo.
The large subunits of the AP complexes contain a number
of other intriguing motifs and domains, including the
“KRIGY” and “WIIGEY” sequences (Hirst et al., 1999).
These motifs are found not only in every member of the
/// and families, but also in the very distantly related
-COP and -COP subunits of the coatomer complex. So
far nothing is known about their function, but we are currently mutating these motifs in both the and 3 subunits.
By taking advantage of the mh and pe cell lines, it should be
possible to assess the importance of these and other sequences not only in the assembly and localization of coat
protein complexes, but also in coated vesicle function.
Materials and methods
Cell lines
Antibody-based methods
Immunoprecipitations were performed both on nondenatured proteins and
in the presence of SDS, using antibodies against both AP-3 and AP-1 subunits, as described previously (Simpson et al., 1996, 1997). Western blotting
and immunofluorescence were also performed using previously published
methods (Robinson and Pearse, 1986; Robinson, 1987). The polyclonal rabbit antibodies against AP-3 subunits have already been described (Simpson
et al., 1996, 1997); in addition, a mouse monoclonal antibody was raised
against the hinge domain (amino acids 608–800) of the AP-3 subunit. The
construct was expressed as a GST fusion protein and used to immunize female BALB/c mice. Fusion of the spleen cells with myeloma cells and
screening of the resulting antibodies were performed as described by Bock
et al. (1997). Ascites fluid was prepared by Joseph Beirao (Josman Laboratories, Napa Valley, CA). The rabbit anticlathrin antibody has been described
previously (Simpson et al., 1997). The monoclonal anti- antibody, mAb
100/3, was purchased from Sigma-Aldrich. The anti–LAMP-1 antibody
(1D4B) was initially obtained from the Developmental Studies Hybridoma
Bank developed under the auspices of the NICHD and maintained by The
University of Iowa, Department of Biological Sciences, Iowa City, IA.
Yeast two-hybrid analysis
Most molecular biology techniques were performed as described by Sambrook and Russell (2001). To construct the plasmids for yeast two-hybrid
analysis, the AP-3 subunits , 3A, 3B, 3A, 3B, 3A, and 3B were all
cloned into the yeast two-hybrid vectors pGBT9 and pGAD424 (CLONTECH Laboratories, Inc.), using PCR to amplify the coding sequences and
to introduce appropriate restriction sites. All constructs that had been
made using PCR were sequenced by John Lester (University of Cambridge,
Cambridge, UK) to confirm that no errors had been introduced. Interactions were monitored by assaying for growth in the absence of histidine.
Expression in mammalian cells
All plasmids for expression in mammalian cells were constructed using the
vector pMEP (Girotti and Banting, 1996). The 3A2 construct consists
of the NH2-terminal 686 amino acids of 3A fused to the last 334 amino
acids of 2. The deletion constructs and the point mutant were made by
PCR and sequenced as above to confirm that they were error-free. Cells
were transfected using QIAGEN SuperFect transfection reagent and selected in DME plus 10% fetal calf serum with Hygromycin (0.2 mg/ml). At
least three cell lines were selected for each construct.
Flow cytometry
A quantitative assay for AP-3 dependent sorting was developed using flow
cytometry. Two rat monoclonal antibodies, anti–LAMP-1 (1D4B) and a
control anti-KLH antibody (both IgG2as) were supplied by BD PharMingen. The antibodies were directly conjugated to Alexa Fluor 488 using the
Molecular Probes Alexa 488 protein-labeling kit according to the manufacturer’s instructions. Cells were then incubated with either anti–LAMP-1
or control antibody for 6 h at 37 in serum-free media. The cells were
rinsed in PBS, trypsinized, fixed in 3% paraformaldehyde, and analyzed
on a fluorescence-activated cell scanner (FACScalibur; Becton Dickinson).
To ensure that differences in anti–LAMP-1 uptake were not due to differences in LAMP-1 expression levels, Western blots of each cell line were
probed with the LAMP-1 antibody. Although there was some variability
from one line to another, there was no correlation between LAMP-1 expression levels and the amount of anti–LAMP-1 uptake.
We are very grateful to Richard Scheller, who generously allowed us to
use an unpublished monoclonal antibody against AP-3 raised in his lab.
We thank Dick Swank for the pearl cell line, Dot Bennett for the melan-a
cell line, and Margit Burmeister for advice on maintaining mocha mice.
We also thank Paul Luzio, Matthew Seaman, David Owen, Rainer Duden,
John Kilmartin, and members of the Robinson lab for reading the manuscript and for helpful discussions.
This work was supported by grants from the Wellcome Trust and the
Medical Research Council.
Submitted: 31 July 2001
Revised: 4 December 2001
Accepted: 4 December 2001
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